Monitoring Reaction Intermediates to Predict Enantioselectivity Using Mass Spectrometry

Abstract Enantioselective reactions are at the core of chemical synthesis. Their development mostly relies on prior knowledge, laborious product analysis and post‐rationalization by theoretical methods. Here, we introduce a simple and fast method to determine enantioselectivities based on mass spectrometry. The method is based on ion mobility separation of diastereomeric intermediates, formed from a chiral catalyst and prochiral reactants, and delayed reactant labeling experiments to link the mass spectra with the reaction kinetics in solution. The data provide rate constants along the reaction paths for the individual diastereomeric intermediates, revealing the origins of enantioselectivity. Using the derived kinetics, the enantioselectivity of the overall reaction can be predicted. Hence, this method can offer a rapid discovery and optimization of enantioselective reactions in the future. We illustrate the method for the addition of cyclopentadiene (CP) to an α,β‐unsaturated aldehyde catalyzed by a diarylprolinol silyl ether.

extracted-ion mobilograms, the NMR sample was also analyzed using ESI-TIMS-TOF-MS after 100-fold dilution in ACN containing 1 M formic acid.

Experimental procedures ESI-TIMS-TOF-MS
Trapped ion mobility spectrometry-mass spectrometry experiments were performed by using a TIMS-TOF MS (Bruker Daltonics Inc., Billerica, MA). Ions were generated by electrospray ionization (ESI), with the following settings: capillary voltage 3.5 kV, end plate offset 500 V, dry gas 3.

NMR experiments
NMR experiments were conducted on a Bruker Avance III 400 MHz or Bruker Avance III 500 MHz spectrometer. Depending on the solvent of the sample, the residual solvent peak of chloroform (δH = 7.26) or formic acid (δH = 8.03) was used as the internal reference.

Helium-tagging photodissociation spectroscopy
Infrared photodissociation spectroscopy (IRPD) experiments were performed by helium-tagging method using the ISORI instrument. [1] The ions were generated under exactly the same conditions as above. The ions of interest were mass-selected and guided with a quadrupole bender and an octopole to a wire quadrupole ion trap operating at 3 K. The ions were trapped using helium buffer gas and after the collisional cooling, the ions formed complexes with helium atoms. The ions in the trap were then irradiated with tunable IR light from OPO. After irradiation, the ions were extracted from the trap, mass-analyzed by a quadrupole and detected by a Daly-type detector. The IRPD spectrum is constructed as 1-Ni()/Nio, where N i () is a number of helium complexes detected after IR irradiation as a wavenumber  and N io is a number of helium complexes without irradiation (measured in alternative cycles with IR light blocked).

Chiral HPLC
Chiral HPLC analysis was performed using a Shimadzu LC-20 HPLC system. To determine the enantiomeric excess of the reaction, aldehydes 6 must have been reduced to the corresponding alcohols 7.
To this end, product 6 was dissolved in isopropanol at a concentration of 4 mg/mL and filtered over a 0.22 µm filter. The sample (5 µL) was injected onto a Phenomenex Lux Cellulose-1 column operated at 35 °C.
Heptane/isopropanol (97:3) was used as eluent at a flow rate of 0.5 ml/min. UV detection at 220 and 254 nm was used to monitor the separation of the R (tr =25.0 min) and S (tr =23.6 min) enantiomer of compound 7.

Computational details
For DFT optimizations, the preferred conformers of all isomers were identified by conformational analysis performed with the PM6 method. The lowest energy conformations were further optimized using the B3LYP functional with the D3 dispersion correction and the 6-31G** basis set as implemented in Gaussian 16. [2] For calculations of IR spectra, scaling factors of 0.985 and 0.952 were used in the region below and above 2000 cm -1 , respectively. Calculations of CCS of DFT-optimized structures were performed by using collidoscope. [3]

Determination of rate constants by fitting data of DRL experiments
Summed rate constants for the depletion of intermediates 3/3' (i.e. k -1 +k 2 ) were obtained by analytical fitting the relative intensity of ion 3' using equation S1 [4] : [3']t = [3']eq (1-e -(k -1 +k 2 )•t ) (S1) In which [3'] t is the relative concentration of 3' (with respect to the sum of 3 and 3') at time 't' and [3'] eq is the relative concentration of 3' after reaching the equilibrium between labeled and unlabeled intermediates. Note that equation 1 is only valid for intermediates displaying steady-state kinetics. To determine separate values for k -1 and k 2 , a DRL experiment was performed in the absence of CP which showed that k -1 is negligible, therefore we used k -1 =0 in further fitting.
For the determination of all other rate constants, the relative ion intensities and isomer ratios were derived using the Euler numerical integration method (see rate equations S1-S6 and the numerical model

Rate equations used for the determination of rate constants
To determine the rate constants of the individual pathways of the asymmetric reaction, rate equations S2-S7 were used. During fitting of the data with the Euler method, the values of k -1 and k 2 were fixed to the outcome obtained via equation 1. Note that in the equations below, the kinetics of the intermediates and product are shown for isomers a as an example. For isomers b and c, individual rate constants were determined using their corresponding concentrations. As CP is added in excess, it is not included in the rate equations.

Synthesis of compound 6
Compound 6 was synthesized based on the procedure reported by Gotoh et al. [5] In a round bottom flask compound 1 (0.4 mmol) and compound 2 (4 mmol) were mixed in 8 mL of methanol.

Characterization of intermediate 3 (IRPD, NMR and DFT)
In order to characterize the structures of the isomers of intermediate 3, we first recorded IRPD spectra in order to investigate whether isomerization of the C=C bond would occur in solution. Hereto, IRPD spectra of two reaction mixtures were recorded: a mixture of catalyst 1 and compound 2 (without CP) in methanol, and a similar mixture containing 100 mM formic acid. From ESI-IM-MS experiments, it was observed that the addition of formic acid promoted the formation of isomer 3a ( Figure S1a), which could be used as a tool for its identification. The IRPD spectra were compared to the theoretical IR spectra of DFT-optimized structures of four potential double C=C-C=N bond isomers (i.e. E-E, E-Z, Z-E and Z-Z). The theoretical spectra are very similar for the iminium ions with E,E and E,Z configurations, but show that isomerization of the C=C bond (to Z-configuration) would result in several shifts in the IR spectrum, especially around 1600 cm -1 ( Figure S1b). Since the IRPD spectra of both reaction mixtures (i.e. with and without formic acid) are essentially identical ( Figure S1c), we concluded that isomerization of the C=C bond does not occur to any notable extent. TOCSY spectra with 1D and 2D NOESY spectra, two isomers of iminium ion 3 could be annotated ( Figure   S2). ESI-IM-MS analysis of the reaction mixture showed a good agreement between the ratio of 3b:3c in the extracted-ion mobilogram and the ratio of the two fully annotated iminium ions in NMR. A third iminium ion was observed as well (δH = 5.11 in Fig S2a), but its relative intensity in the NMR spectrum did not match with the relative intensity of 3a in the mobilogram. After a prolonged reaction time (6 days total reaction time), this third iminium ion was substantially increased in abundance. As none of the integrals of the chemical shifts around 0 ppm (i.e. TMS protons) matched with a 9-fold of the integrals of the protons of the third isomer, we concluded that the third iminium ion in the NMR spectrum corresponds to a structure with a hydrolyzed TMS ether, rather than an isomer of 3b and 3c. respectively. The insert shows the extracted-ion mobilogram (m/z 470) of the 2 days incubated sample recorded using ESI-IM-MS after dilution. The numbers above the peaks indicate the integrals relative to the largest peak. b&c, Summary of the NMR characterization of two iminium isomers that were annotated as ion 3c and 3b. As can be observed in (a), the relative intensities of their NMR signals are in good agreement with those in the extracted-ion mobilogram. Note that the isomer 3a was impossible to detect by NMR, the third detected species with the increasing relative intensity with time (blue vs. red) is an iminium ion with hydrolyzed TMS ether. d, Summary of the NMR characterization of a third iminium ion (with hydrolyzed TMS ether). NOESY and COSY correlations of geminal protons are not shown, and integrals are only displayed for protons showing no or very limited overlap in the 1H NMR spectrum. It should be noted that some expected correlations (especially for COSY) were not detected due to the limited intensity of signals. NMR spectra used for the annotation are shown in Figure S15-S32.

Experiments on the mass spectrometric detection of intermediate 4
The As can be observed from Table S2, the relative energy of N-protonated enamines exceeds their Cprotonated analogues by >10 kcal/mol. This suggests that C-protonation occurs exclusively upon ESI of enamines 4. A comparison between the theoretical IR spectra and experimental IRPD spectrum of m/z 536 ( Figure S3) also indicates that no N-protonated enamines are detected in the ESI-MS experiments. Thus, two possible scenarios remain: i) enamines 4 are rapidly protonated to iminium ions 5 in solution or ii) a mixture of intermediates 4 and 5 is present in solution, but both intermediates are detected as iminium ions 5 upon ESI. As the former hypothesis seems to be incompatible with obtaining a good fit in the modelling of DRL experiments, we conclude that the latter (i.e. conversion of 4 into 5 during ESI) occurs in practice.  In addition, relatively small intensities of m/z 398 and 401 were detected. These m/z values correspond to iminium ions formed between reactant 2(') and catalyst 1 of which the TMS ether has been hydrolyzed. In the mass spectrum recorded with the tims-cell disabled, an ion with m/z 254 is detected (loss of 72 Da compared to the intact catalyst), confirming that the hydrolyzed catalyst is indeed present. Hydrolysis of TMS ethers in Jørgensen-Hayashi type catalysts has been reported previously by others. [6] The iminium ions formed via the free -OH catalysts have been suggested to exist in equilibrium with oxazolidine species, via intramolecular cyclization, resulting in a diminished reactivity. [7] We are currently investigating this phenomenon and the effect on the kinetics of the intermediates in more detail, using the novel method presented in this study.

S13
Analytical fitting of k -1 + k 2 Figure S4 Ratios of 3c/3c' (a&b), 3b/3b' (c&d), and 3a/3a' (e&f) in two (duplicate) delayed reactant labeling experiments. Relative intensities of labeled and unlabeled intermediates are shown in red and black, respectively. The black lines represent the fits of 3c' according to equation 1 (see method section). The values of a and b in the boxes represent the fitted relative concentrations of the labeled ions at equilibrium, and the summed rate constants of the depletion of the intermediates (i.e. k-1 + k2).

Figure S5 TIC-normalized extracted ion mobilograms illustrating the time evolution of individual intermediate isomers for 3 & 3'
in a DRL experiment without CP. The equilibrium formation between labeled and unlabeled species is slow in comparison to the situation with CP (see Figure 4). The slower depletion of the iminium intermediates in absence of CP indicates that the rate of hydrolysis of intermediate 3 (to the reactant and catalyst) is slow in comparison to the nucleophilic attack by CP. Interestingly, the relative abundance of isomer a seems to increase over time. This might indicate slow isomerization of between the intermediate 3 isomers. As this process is slow, it is not observed in presence of CP, as all isomers rapidly react with CP.   Table 1 (d-f and j-l). The predicted responses (Y-axes) in d-f and j-l were calculated by summing the predicted concentrations of ions 4 and 5 with a correction factor of 0.025 for ion 4 to account for its lower ionization efficiency.

Figure S9
Predicted percentage of the major (R) enantiomer for various catalyst loadings at 45 °C. The solid lines were constructed using the average rate constants of the two duplicate experiments (see Table 1 and Table S3). For the construction of the dashed lines, k4 of the major pathway was lowered to the average of k4 of the minor pathway (0.06 min -1 ). The graph illustrates that k4 has no effect on the enantiomer ratio at full conversion, and only a small effect at low conversions. *Note that for the calculation of the enantiomer ratio, we assume that intermediates 4 and 5 are eventually fully hydrolyzed to product 6 (i.e. the Y-axis corresponds to the sum of 4c, 5c and R-6).

Duplicate of delayed reactant labeling experiment at 45°C
In order to check the reproducibility of the novel method, a duplicate of the DRL experiment was performed. Comparing Figure S10 with Figure 5 and Table 1 with Table S3 indicates that only small deviations are observed between the two duplicate experiments.  Table S3 Table S4  The blue curves were constructed by summing the concentrations of enamine 4 and iminium ion 5, using a correction factor of 0.04 to account for the lower ionization efficiency of the enamine.  For the main product pathway (3C4/5B), the input distribution of the kinetic parameters for MC simulation is shown in Figure S12b. The kinetic parameters with high variances are k 3 and k 4 , where the high variance of the former is caused by the indefinite in the starting point of the experiment (i.e. t d ), while the latter is due to significantly lowered concentration in the process of asymmetric reaction. MC sampled points were observed to give significantly higher absolute residual than the fitted kinetic model using sequential MC simulation for kinetic parameters (see Fig S12c). The implication is that further tuning of the kinetic parameters will not significantly improve the model's statistical performance (z = 0.0944, p<0.001). Further analysis using kernel density estimation of the principal components of the kinetic parameters ( Fig S12d) shows that the fitted model has been densely search around its local space, ensuring that the model is situated within an optimal point.  Figure   S12e), it can be derived that the rate constants of the forward reaction are more important than those corresponding to the reverse reaction. An additional MC simulation was carried out on the initial experimental conditions (i.e. concentrations) while analyzed with the product yields in a principal component analysis scores projected on loadings. In Figure S12f, the initial experimental conditions for compound 1, 1' and 2 are represented in the PC2 axis, while the product yield (major, minor and side products) was represented in the PC1 axis. The clump of dark green points in the middle with stationary value in PC 1 with majority of MC sampled points imply that initial conditions were not significantly affecting the product yields of the asymmetric reaction. This demonstrates that uncertainty in initial experimental conditions does not significantly impact the model prediction. The inverse direction of the loading arrows for major product with respect to minor and side product suggests a competing mechanism between product pathways.
Using the same method as Monte Carlo simulation 1, two additional analyses were performed with slightly different sets of restrictions (see captions of Table S5-S6). The starting points of the Monte-Carlo simulations was determined using analytical fitting. From the results (Table S5-S6 and Figure S13-S14) it can be observed that the k -1 parameter within the reaction system is not crucial in giving accurate estimation of relative intensities over time. This confirms the findings from Figure S12e, showing that k-1 has low importance in characterizing the asymmetric reaction system. The simulations also demonstrate that the fundamental kinetic parameters that characterizes the asymmetric reaction system is mainly k 2 and k 3 .

Table S5
Rate constants refined using sequential Monte-Carlo simulations, with the following restrictions: k -1 fixed at 0.025, k 2 fixed to values of Table 1, k -2 and k -3 fixed at 0. Other rate constants were optimized by the sequential Monte-Carlo simulation.  Table S5

Table S6
Rate constants refined using sequential Monte-Carlo simulations, with the following restrictions: k1 and k2 fixed to values of Table 1, k -1 , k -2 and k -3 fixed at 0. Other rate constants were optimized by the sequential Monte-Carlo simulation.